Assays for sensory modulators using a sensory cell specific G-protein alpha subunit

ABSTRACT

The invention identifies nucleic acid and amino acid sequences of a sensory cell specific G-protein alpha subunit that are specifically expressed in sensory cells, e.g., taste cells, antibodies to such G-protein alpha subunits, methods of detecting such nucleic acids and subunits, and methods of screening for modulators of a sensory cell specific G-protein alpha subunit.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/117,367, filed Jan.27, 1999, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. DC03160,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The invention identifies nucleic acid and amino acid sequences of ataste cell specific G-protein alpha subunit that are specificallyexpressed in taste cells, antibodies to such G-protein alpha subunits,methods of detecting such nucleic acids and subunits, and methods ofscreening for modulators of taste cell specific G-protein alpha subunit.

BACKGROUND OF THE INVENTION

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (see, e.g., Avenet & Lindemann, J. MembraneBiol. 112:1-8 (1989); Margolskee, BioEssays 15:645-650 (1993)).Gustatory signaling is found throughout the animal kingdom, from simplemetazoans to the most complex of vertebrates; its main purpose is toprovide a reliable signaling response to non-volatile ligands. Higherorganisms have four basic types of taste modalities: salty, sour, sweet,and bitter. Each of these modalities is thought to be mediated bydistinct signaling pathways leading to receptor cell depolarization,generation of a receptor or action potential, and the release ofneurotransmitter and synaptic activity (see, e.g., Roper, Ann. Rev.Neurosci. 12:329-353 (1989)).

Mammals are believed to have five basic taste modalities: sweet, bitter,sour, salty and unami (the taste of monosodium glutamate) (see, e.g.,Kawamura & Kare, Introduction to Umami: A Basic Taste (1987); Kinnamon &Cummings, Ann. Rev. Physiol. 54:715-731(1992); Lindemann, Physiol. Rev.76:718-766 (1996); Stewart et al., Am. J. Physiol. 272:1-26 (1997)).Extensive psychophysical studies in humans have reported that differentregions of the tongue display different gustatory preferences (see,e.g., Hoffmann, Menchen. Arch. Path. Anat. Physiol. 62:516-530 (1875);Bradley et al., Anatomical Record 212:246-249 (1985); Miller & Reedy,Physiol. Behav. 47:1213-1219 (1990)). Also, numerous physiologicalstudies in animals have shown that taste receptor cells may selectivelyrespond to different tastants (see, e.g., Akabas et al., Science242:1047-1050 (1988); Gilbertson et al., J. Gen. Physiol. 100:803-24(1992); Bernhardt et al., J. Physiol. 490:325-336 (1996); Cummings etal., J. Neurophysiol. 75:1256-1263 (1996)). In mammals, taste receptorcells are assembled into taste buds that are distributed into differentpapillae in the tongue epithelium. Circumvallate papillae, found at thevery back of the tongue, contain hundreds (mice) to thousands (human) oftaste buds and are particularly sensitive to bitter substances. Foliatepapillae, localized to the posterior lateral edge of the tongue, containdozens to hundreds of taste buds and are particularly sensitive to sourand bitter substances. Fungiform papillae containing a single or a fewtaste buds are at the front of the tongue and are thought to mediatemuch of the sweet taste modality.

Each taste bud, depending on the species, contain 50-150 cells,including precursor cells, support cells, and taste receptor cells (see,e.g., Lindemann, Physiol. Rev. 76:718-766 (1996)). Receptor cells areinnervated at their base by afferent nerve endings that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. Elucidating the mechanisms of taste cellsignaling and information processing are critical for understanding thefunction, regulation, and “perception” of the sense of taste.

Although much is known about the psychophysics and physiology of tastecell function, very little is known about the molecules and pathwaysthat mediate these sensory signaling responses (reviewed by Gilbertson,Current Opn. in Neurobiol. 3:532-539 (1993); see also McLaughlin et al.,Nature 357:563-568 (1992)). Electrophysiological studies suggest thatsour and salty tastants modulate taste cell function by direct entry ofH⁺ and Na⁺ ions through specialized membrane channels on the apicalsurface of the cell. In the case of sour compounds, taste celldepolarization is hypothesized to result from H⁺ blockage of K⁺ channels(see, e.g., Kinnamon et al., PNAS USA 85:7023-7027 (1988)) or activationof pH-sensitive channels (see, e.g., Gilbertson et al., J. Gen. Physiol.100:803-24 (1992)); salt transduction may be partly mediated by theentry of Na⁺ via amiloride-sensitive Na⁺ channels (see, e.g., Heck etal., Science 223:403-405 (1984); Brand et al., Brain Res. 207-214(1985); Avenet et al., Nature 331:351-354 (1988)). Most of molecularcomponents of the sour or salty pathways have not been identified.

Sweet, bitter, and unami transduction are believed to be mediated byG-protein-coupled receptor (GPCR) signaling pathways (see, e.g., Striemet al., Biochem. J. 260:121-126 (1989); Chaudhari et al., J. Neuros.16:3817-3826 (1996); Wong et al., Nature 381:796-800 (1996)).Confusingly, there are almost as many models of signaling pathways forsweet and bitter transduction as there are effector enzymes for GPCRcascades (e.g., G protein subunits, cGMP phosphodiesterase,phospholipase C, adenylate cyclase; see, e.g., Kinnamon & Margolskee,Curr. Opin. Neurobiol 6:506-513 (1996)). Identification of moleculesinvolved in taste signaling is important given the numerouspharmacological and food industry applications for bitter antagonists,sweet agonists, and modulators of salty and sour taste.

The identification and isolation of taste receptors (including taste ionchannels), and taste signaling molecules, such as G-protein subunits andenzymes involved in signal transduction, would allow for thepharmacological and genetic modulation of taste transduction pathways.For example, availability of receptor, ion channels, and other moleculesinvolved in taste transduction would permit the screening for highaffinity agonists, antagonists, inverse agonists, and modulators oftaste cell activity. Such taste modulating compounds could then be usedin the pharmaceutical and food industries to customize taste. Inaddition, such taste cell specific molecules can serve as invaluabletools in the generation of taste topographic maps that elucidate therelationship between the taste cells of the tongue and taste sensoryneurons leading to taste centers in the brain.

SUMMARY OF THE INVENTION

The present invention demonstrates, for the first time, taste receptorcell specific expression of nucleic acids encoding G-protein alphasubunit. Specifically, the present invention identifies that G14, aG-protein alpha subunit, is specifically and selectively expressed intaste receptor cells. This gene was found to be co-expressed withG-protein coupled taste receptors, GPCR-B3 and GPCR-B4 (see U.S. Ser.No. 09/361,652, filed Jul. 27, 1999 and U.S. Ser. No. 09/361,631, filedJul. 27, 1999, now U.S. Pat. No. 6,383,778). Functionally, GPCR-B3 andGPCR-B4 each represents a seven transmembrane G-protein coupled receptorinvolved in taste transduction, which interacts with a G-protein tomediate taste signal transduction (see e.g., Fong, Cell Signal8:217(1996): Baldwin. Curr. Opin. Cell Biol. 6:180 (1994)).Structurally, exemplary polynucleotide sequences encoding rat, mouse,and human GPCR-B3 are presented in SEQ ID NOs:3-5, respectively,encoding polypeptides having the amino acid sequences of SEQ ID NOs:6-8.Exemplary polynucleotide sequences encoding rat, mouse, and humanGPCR-B4 are presented in SEQ ID NOs:9-11, respectively, encodingpolypeptides having the amino acid sequences of SEQ ID NOs: 12-14. Thesetaste receptors have been previously shown to be expressed intopographically distinct subpopulations of taste receptor cells andtaste buds. These receptors are specifically localized to the tastepore, and are distantly related to putative mammalian pheromonereceptors. The present invention thus demonstrates that Gα14 isspecifically expressed in taste cells and further that it isco-expressed with GPCR-B3 and GPCR-B4 receptors in the different tastepapillae. The G-protein alpha subunits that are specifically expressedin taste cells can thus be used, e.g., to screen for modulators oftaste. The compounds identified by these assays would then be used bythe food and pharmaceutical industries to customize taste, e.g., asadditives to food or medicine so that the food or medicine tastesdifferent to the subject who ingests it. For example, bitter medicinescan be made to taste less bitter, and sweet substance can be enhanced.

In one aspect, the present invention provides a method for identifying acompound that modulates sensory signaling in sensory cells, the methodcomprising the steps of: (i) contacting the compound with a sensory cellspecific G-protein alpha subunit polypeptide, the G-protein alphasubunit polypeptide comprising greater than about 70% amino acidsequence identity to a polypeptide having a sequence of SEQ ID NO:2; and(ii) determining a functional effect of the compound upon the G-proteinalpha subunit polypeptide.

In one embodiment, the G-protein alpha subunit polypeptide specificallybinds to polyclonal antibodies generated against SEQ ID NO:2. In anotherembodiment, the G-protein alpha subunit polypeptide is recombinant. Inanother embodiment, the G-protein alpha subunit polypeptide is from amouse, a rat or a human. In another embodiment, the G-protein alphasubunit polypeptide comprises an amino acid sequence of SEQ ID NO:2. Inanother embodiment, the G-protein alpha subunit polypeptide is linked toa solid phase, either covalently or non-covalently. In anotherembodiment, the G-protein alpha subunit is a domain of a G-protein alphasubunit comprising greater than about 70% identity to a G-protein alphasubunit domain of a polypeptide having the amino acid sequence of SEQ IDNO:2. In another embodiment, the G-protein alpha subunit is a fusionpolypeptide. In another embodiment, the G-protein alpha subunit is adomain fused to a heterologous polypeptide to form a fusion polypeptide.

In one embodiment, the functional effect is a chemical effect or aphysical effect. In another embodiment, the functional effect isdetermined by measuring binding of radiolabeled GTP to the G-proteinalpha subunit polypeptide or to a G protein comprising the G-proteinalpha subunit polypeptide.

In one embodiment, the G-protein alpha subunit polypeptide is expressedin a cell or a cell membrane. In another embodiment, the cell or cellmembrane is attached to a solid substrate. In another embodiment, thecell is a eukaryotic cell, e.g., a human cell, e.g., an HEK 293 cell. Inanother embodiment, the G-protein alpha subunit polypeptide isco-expressed with GPCR-B3 or GPCR-B4.

In one embodiment, the functional effect is determined by measuringchanges in intracellular cAMP, cGMP, IP₃, DAG, or intracellular Ca²⁺,e.g., using immunoassays. In another embodiment, the functional effectis measured by determining changes in the electrical activity of cellsexpressing the G-protein alpha subunit polypeptide, e.g., with an assayselected from the group consisting of a voltage clamp assay, a patchclamp assay, a radiolabeled ion flux assay, or a fluorescence assayusing voltage sensitive dyes. In another embodiment, the functionaleffect is determined by measuring changes in the level ofphosphorylation of sensory cell specific proteins. In anotherembodiment, the functional effect is determined by measuring changes intranscription levels of sensory cell specific genes.

In another aspect, the present invention provides a method foridentifying a compound that modulates sensory signaling in sensorycells, the method comprising the steps of: (i) expressing a sensory cellspecific G-protein alpha subunit polypeptide in an HEK 293 host cell,wherein the G-protein alpha subunit polypeptide comprises greater thanabout 70% amino acid sequence identity to a polypeptide having asequence of SEQ ID NO:2; (ii) expressing a sensory cell specificG-protein coupled receptor in the host cell; (iii) contacting the hostcell with the compound that modulates sensory signaling in sensorycells; and (iv) determining changes in intracellular calcium levels inthe host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention demonstrates that nucleic acids encoding G-proteinalpha 14 subunit are specifically expressed in taste cells. Thesenucleic acids and the polypeptides that they encode are referred to as“TC-Gα14” for “taste cell specific G-protein alpha 14 subunit.” Thesetaste cell specific nucleic acids and polypeptides are components of thetaste transduction pathway and are G-protein alpha subunits involved intaste transduction.

The invention thus provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of TC-Gα14. Such modulators of taste transduction areuseful for pharmacological and genetic modulation of taste signalingpathways. These methods of screening can be used to identify highaffinity agonists and antagonists of taste cell activity. Thesemodulatory compounds can then be used in the food and pharmaceuticalindustries to customize taste. For example, the modulatory compoundswould be added to a food or medicine, thereby altering its taste to thesubject who ingests it.

Thus, the invention provides assays for taste modulation, where TC-Gα14acts as a direct or indirect reporter molecule for the effect ofmodulators on taste transduction. TC-Gα14 can be used in assays, e.g.,to measure changes in ion concentration, membrane potential, currentflow, ion flux, transcription, signal transduction, receptor-ligandinteractions, G-protein binding to receptors; binding to other G proteinbeta and gamma subunits; binding to enzymes;; G-protein subunit ligandbinding; second messenger concentrations, in vitro, in vivo, and exvivo. In one embodiment, TC-Gα14 is recombinantly expressed in cells,optionally with taste specific GPCR such as GPCR-B3 or GPCR-B4, andmodulation of taste transduction is assayed by measuring changes in Ca²⁺levels (see Example II). In another embodiment, binding of radiolabeledGTP to TC-Gα14 or a G-protein comprising TC-Gα14 is measured,.

Methods of assaying for modulators of taste transduction include oocyteor tissue culture cell expression of TC-Gα14; transcriptional activationof TC-Gα14; phosphorylation and dephosphorylation of TC-Gα14; ligandbinding assays; voltage, membrane potential and conductance changes; ionflux assays; changes in intracellular second messengers such as cAMP,cGOMP, and inositol triphosphate; changes in intracellular calciumlevels; and neurotransmitter release.

Finally, the invention provides for methods of detecting TC-Gα14 nucleicacid and protein expression, allowing investigation of tastetransduction regulation and specific identification of taste receptorcells, as the nucleic acids are specifically expressed in taste cells.For example, probes for TC-Gα14 can be used to identify subsets of tastecells such as foliate cells and circumvallate cells, or specific tastereceptor cells, e.g., sweet, sour, salty, and bitter. TC-Gα14polypeptides can also be used to generate monoclonal and polyclonalantibodies useful for identifying taste receptor cells, e.g., in immunohistochemical assays. Taste receptor cells can be identified usingtechniques such as reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, S1 digestion, high densityoligonucleotide arrays, western blots, and the like. The nucleic acidsand the proteins that they encode also serve as tools for the generationof taste topographic maps that elucidate the relationship between thetaste cells of the tongue and taste sensory neurons leading to tastecenters in the brain. Furthermore, the nucleic acids and the proteinsthey encode can be used as probes to dissect taste-induced behaviors.TC-Gα14 also provides useful nucleic acid probes for paternity andforensic investigations.

Functionally, TC-Gα14 represents an alpha subunit of a heterotrimericG-protein, which interacts with a GPCR to mediate taste signaltransduction (see, e.g., Fong, Cell Signal 8:217 (1996); Baldwin, Curr.Opin. Cell Biol. 6:180 (1994)). G-proteins are composed of alpha, beta,and gamma subunits. The alpha subunit of a G-protein binds guaninenucleotide and is believed to confer receptor and effector specificity.G proteins mediate the interaction between G protein coupled receptorsand signal transduction enzymes such as adenylate cyclase andphospholipase C.

Structurally, the nucleotide sequence of TC-Gα14 (see, e.g., SEQ ID NO:1isolated from mouse) encodes a polypeptide of approximately 355 aminoacids with a predicted molecular weight of approximately 41 kDa and apredicted range of 36-46 kDa (see, e.g., the amino acid sequence of Ga14 published in Wilkie et al., PNAS USA 88:10049-10053 (1991), SEQ IDNO:2). Related TC-Gα14 genes from other species share at least about 70%amino acid identity over an amino acid region at least about 25 aminoacids in length, preferably 50 to 100 amino acids in length. TC-Gα14 isspecifically expressed in taste receptor cells.

The present invention also provides polymorphic variants of the TC-Gα14depicted in SEQ ID NO:2: variant #1, in which an isoleucine residue issubstituted for the leucine residue at amino acid position 7; variant#2, in which an aspartic acid residue is substituted for the glutamicacid residue at amino acid position number 20; an variant #3, in whichan alanine residue is substituted for the glycine residue at amino acidposition 60.

Specific regions of the TC-Gα14 nucleotide and amino acid sequences maybe used to identify polymorphic variants, interspecies homologs, andalleles of TC-Gα14. This identification can be made in vitro, e.g.,under stringent hybridization conditions or with PCR and sequencing, orby using the sequence information in a computer system for comparisonwith other nucleotide or amino acid sequences. Typically, identificationof polymorphic variants and alleles of TC-Gα14 is made by comparing anamino acid sequence of about 25 amino acids or more, preferably 50-100amino acids. Amino acid identity of approximately at least about 70% orabove, preferably 80%, most preferably 90-95% or above typicallydemonstrates that a protein is a polymorphic variant, interspecieshomolog, or allele of TC-Gα14. Sequence comparison can be performedusing any of the sequence comparison algorithms discussed below,preferably with the BLAST or BLAST 2.0 algorithm with defaultparameters, and either the BLASTN or BLASTP program with defaultparameters, as discussed below. Antibodies that bind specifically toTC-Gα14 or a conserved region thereof can also be used to identifyalleles, interspecies homologs, and polymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of TC-Gα14 areconfirmed by examining taste cell specific expression of the putativeTC-Gα14 polypeptide. Typically, TC-Gα14 having the amino acid sequenceof SEQ ID NO:2 is used as a positive control, e.g., in immunoassaysusing antibodies directed against the amino acid sequence of SEQ IDNO:2, in comparison to the putative TC-Gα14 protein to demonstrate theidentification of a polymorphic variant or allele of TC-Gα14.Alternatively, TC-Gα14 having the nucleic acid sequences of SEQ ID NO: 1is used as a positive control, e.g., in in situ hybridization with SEQID NO: 1, in comparison to the putative TC-Gα14 nucleotide sequences todemonstrate the identification of a polymorphic variant or allele ofTC-Gα14. The polymorphic variants, alleles and interspecies homologs ofTC-Gα14 are expected to retain the ability to form a heterotrimericG-protein.

TC-Gα14 nucleotide and amino acid sequence information may also be usedto construct models of taste cell specific polypeptides in a computersystem. These models are subsequently used to identify compounds thatcan activate or inhibit TC-Gα14. Such compounds that modulate theactivity of TC-Gα14 can be used to investigate the role of TC-Gα14 intaste transduction or can be used as therapeutics.

Identification of taste cell specific expression of TC-Gα14 provides forthe first time a means for assaying for inhibitors and activators oftaste cell activity. TC-Gα14 is useful for testing taste modulatorsusing in vivo and in vitro expression that measure, e.g.,transcriptional activation of TC-Gα14; ligand binding; ligand binding(e.g., radiolabeled GTP biding to TC-Gα14 or a G-protein comprisingTC-Gα14); phosphorylation and dephosphorylation; binding to G-proteins;G-protein activation; regulatory molecule binding; voltage, membranepotential and conductance changes; ion flux; intracellular secondmessengers such as cAMP, cGMP, and inositol triphosphate; intracellularcalcium levels; and neurotransmitter release. Such activators andinhibitors identified using TC-Gα14 can be used to further study tastetransduction and to identify specific taste agonists and antagonists.Such activators and inhibitors are useful as pharmaceutical and foodagents for customizing taste.

Methods of detecting TC-Gα14 nucleic acids and expression of TC-Gα14 arealso useful for identifying taste cells and creating topological maps ofthe tongue and the relation of tongue taste receptor cells to tastesensory neurons in the brain. Furthermore, these nucleic acids can beused to diagnose diseases related to taste by using assays such asnorthern blotting, dot blotting, in situ hybridization, RNaseprotection, and the like. Chromosome localization of the genes encodinghuman TC-Gα14 can be used to identify diseases, mutations, and traitscaused by and associated with TC-Gα14. Techniques, such as high densityoligonucleotide arrays (GeneChip™), can be used to screen for mutations,polymorphic variants, alleles and interspecies homologs of TC-Gα14.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Sensory cells” are cells that are found in sensory organs or partsthereof (e.g., taste buds, retina, olfactory epithelium, etc.) and thatparticipate in sensing an external stimulus.

“Sensory cell specific” genes or proteins refer to those which areexpressed exclusively, or preferentially, in the sensory cells but notin non-sensory cells.

“Taste cells” are neuroepithelial cells that are organized into groupsto form taste buds of the tongue, e.g., foliate, fungiform, andcircumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329-353 (1989)). Taste cells also include cells of the palate, andother tissues that may contain taste cells such as the esophagus and thestomach.

“Taste cell specific” genes or proteins refer to those which areexpressed exclusively, or preferentially, in the taste cells but not innon-taste cells, or in subsets of Gustducin positive cells.

“Taste cell specific G-protein alpha subunit” or “TC-Gα14” refers to aG-protein alpha subunit that is specifically expressed in taste receptorcells such as foliate, fungiform, and circumvallate cells. Such tastecells can be identified because they express molecules such asGustducin, a taste cell specific G-protein (McLaughin et al., Nature357:563-569 (1992)). Taste receptor cells can also be identified on thebasis of morphology (see, e.g., Roper, supra). TC-Gα encodes a G-proteinalpha subunit with the ability to form a subunit of a heterotrimericG-protein, that has “G-protein subunit activity,” e.g., has the abilityto form G-proteins that bind GTP. The alpha subunit binds guaninenucleotide and is believed to confer receptor and effector specificity.In response to extracellular stimuli, G-protein coupled receptors bindto G-proteins and promote production of second messengers such as IP₃,cAMP, and Ca²⁺ via stimulation of enzymes such as phospholipase C andadenylate cyclase (for description of the structure and function ofG-proteins and G-protein coupled receptors, see, e.g., Fong, supra,Baldwin, supra, McLaughlin, supra, Wilkie et al. PNAS USA 88:10049-10053(1991)).

Protein “domains” such as a ligand binding domain, an active site, asubunit association region, etc. are found in the polypeptides of theinvention. Such domains are useful for making chimeric proteins and forin vitro assays of the invention. These domains can be structurallyidentified using methods known to those of skill in the art, such assequence analysis programs that identify hydrophobic and hydrophilicdomains (see, e.g., Kyte & Doolittle, J. Mol. Biol. 157:105-132 (1982)).

A “TC-Gα14 domain” refers to a ligand binding domain, a subunitassociation domain, an active site, etc, identified as described above,that has at least about 70% identity to a a ligand binding domain, asubunit association domain, an active site, etc. from a polypeptidehaving a sequence of SEQ ID NO:2. Such domains can be used to makerecombinant fusion proteins or chimeras, where a TC-Gα14 domain is fusedto another molecule, such as a reporter molecule, e.g., GreenFluorescent Protein, β-gal, etc. Fusion proteins can also be made usinga full length TC-Gα14 polypeptide.

The term “TC-Gα14” therefore refers to polymorphic variants, alleles,mutants, and interspecies homologs and TC-Gα14 domains thereof that: (1)have about 70% amino acid sequence identity, preferably about 75, 80,85, 90 or 95% or higher amino acid sequence identity to SEQ ID NO:2 overa window of about 25 amino acids, preferably 50-100 amino acids; (2)bind to antibodies raised against an immunogen comprising an amino acidsequence of SEQ ID NO:2 and conservatively modified variants thereof; or(3) specifically hybridize (with a size of at least about 500,preferably at least about 900 nucleotides) under stringent hybridizationconditions to a sequence SEQ ID NO:1, and conservatively modifiedvariants thereof. This term also refers to a domain of TC-Gα14 asdescribed above.

“TC-GPCR” refers to a G-protein coupled receptor that is specificallyexpressed in taste receptor cells such as foliate, fungiform, andcircumvallate cells. Such taste cells can be identified because theyexpress molecules such as Gustducin, a taste cell specific G-protein(McLaughin et al., Nature 357:563-569 (1992)). Taste receptor cells canalso be identified on the basis of morphology (see, e.g., Roper, supra).Examples of TC-GPCR include GPCR-B3 and GPCR-B4 (see, e.g., Hoon et al.,Cell 96:541-551 (1999); see also U.S. Ser. No. 09/361,652, filed Jul.27, 1999 and U.S. Ser. No. 09/361,631, filed Jul. 27, 1999, now U.S.Pat. No. 6,383,778), herein incorporated by reference in theirentirety). Exemplary polynucleotide sequences encoding rat, mouse, andhuman GPCR-B3 are presented in SEQ ID NOs:3-5, respectively, encodingpolypeptides having the amino acid sequences of SEQ ID NOs:6-8.Exemplary polynucleotide sequences encoding rat, mouse, and humanGPCR-B4 are presented in SEQ ID NOs:9-11, respectively, encodingpolypeptides having the amino acid sequences of SEQ ID NOs: 12-14.TC-GPCRs encode G-protein coupled receptors with seven transmembraneregions that have “G-protein coupled receptor activity,” as describedbelow, e.g., they bind to G-proteins in response to extracellularstimuli and promote production of second messengers such as IP₃, cAMP,and Ca²⁺ via stimulation of enzymes such as phospholipase C andadenylate cyclase (for a description of the structure and function ofG-protein coupled receptors, see, e.g., Fong, supra, and Baldwin,supra).

“GPCR activity” refers to the ability of a GPCR to transduce a signal.Such activity can be measured in a heterologous cell, by coupling a GPCR(or a chimeric GPCR) to either an endogenous G-protein, a promiscuousG-protein subunit such as Gα15, or a taste specific G-protein subunitsuch as TC-Gα14, and an enzyme such as PLC, and measuring increases inintracellular calcium using (Offermans & Simon, J. Biol. Chem.270:15175-15180 (1995)). Receptor activity can be effectively measuredby recording ligand-induced changes in [Ca²⁺]_(i) using fluorescentCa²⁺-indicator dyes and fluorometric imaging. Optionally, thepolypeptides of the invention are involved in sensory transduction,optionally taste transduction in taste cells.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, HEK 293 and the like.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides of TC-Gα14. Suchsamples include, but are not limited to, tissue isolated from humans,mice, and rats, in particular, tongue. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes. A biological sample is typically obtained from aeukaryotic organism, such as insects, protozoa, birds, fish, reptiles,and preferably a mammal such as rat, mouse, cow, dog, guinea pig, orrabbit, and most preferably a primate such as chimpanzees or humans.Preferred tissues include tongue tissue and isolated taste buds.

The phrase “functional effects” in the context of assays for testingcompounds that modulate TC-Gα14 mediated taste transduction includes thedetermination of any parameter that is indirectly or directly under theinfluence of TC-Gα14 or a G-protein comprising TC-Gα14, e.g., afunctional, physical, or chemical effect. It includes ligand binding,changes in ion flux, membrane potential, current flow, radiolabled GTPbinding, subunit association, transcription, G-protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP₃, or intracellular Ca²⁺), in vitro, in vivo, and ex vivoand also includes other physiologic effects such increases or decreasesof neurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of TC-Gα14, e.g., functional, physical and chemicaleffects. Such functional effects can be measured by any means known tothose skilled in the art, e.g., changes in spectroscopic characteristics(e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g.,shape), chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, radiolabeled GTP binding, oocyte TC-Gα14 expression;tissue culture cell TC-Gα14 expression; transcriptional activation ofTC-Gα14; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP and inositol triphosphate (IP3); changes inintracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of TC-Gα14 are usedinterchangeably to refer to inhibitory, activating, or modulatingmolecules identified using in vitro and in vivo assays for tastetransduction, e.g., ligands, agonists, antagonists, and their homologsand mimetics. Inhibitors are compounds that, e.g., bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate taste transduction, e.g.,antagonists. Activators are compounds that, e.g., bind to, stimulate,increase, open, activate, facilitate, enhance activation, sensitize orup regulate taste transduction, e.g., agonists. Modulators includecompounds that, e.g., alter the interaction of a polypeptide with: Gprotein coupled receptors; extracellular proteins that bind activatorsor inhibitor (e.g., ebnerin and other members of the hydrophobic carrierfamily); G-proteins; G protein alpha and beta subunits; kinases (e.g.,homologs of rhodopsin kinase and beta adrenergic receptor kinases thatare involved in deactivation and desensitization of a receptor); andarrestin-like proteins, which also deactivate and desensitize receptors.Modulators include genetically modified versions of TC-Gα14, e.g., withaltered activity, as well as naturally occurring and synthetic ligands,antagonists, agonists, small chemical molecules and the like. Suchassays for inhibitors and activators include, e.g., expressing TC-Gα14in vitro, in cells, or cell membranes, applying putative modulatorcompounds, and then determining the functional effects on tastetransduction, as described above.

Samples or assays comprising TC-Gα14 that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofinhibition. Control samples (untreated with inhibitors) are assigned arelative TC-Gα14 activity value of 100%. Inhibition of TC-Gα14 isachieved when the TC-Gα14 activity value relative to the control isabout 80%, preferably 50%, more preferably 25-0%. Activation of TC-Gα14is achieved when the TC-Gα14 activity value relative to the control(untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolatedTC-Gα14 nucleic acid is separated from open reading frames that flankthe TC-Gα14 gene and encode proteins other than TC-Gα14. The term“purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure.

“Biologically active” TC-Gα14 refers to TC-Gα14 having tastetransduction activity in taste receptor cells or in an assay system withadditional signal transduction components of the taste transductionsystem.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins    (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which ant or 7 can bemade detectable, e.g., by incorporating a radiolabel into the peptide,and used to detect antibodies specifically reactive with the peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity over a specified region (see, e.g., SEQ ID NO:2)), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testsequence. Preferably, the identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately. stringent hybridizationconditions. Exemplary “moderately stringent hybridization conditions”include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDSat 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is atleast twice background. Those of ordinary skill will readily recognizethat alternative hybridization and wash conditions can be utilized toprovide conditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-TC-Gα14” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the TC-Gα14 gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to TC-Gα14 from specific species such as rat, mouse, or human canbe selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with TC-Gα14, and not with other proteins,except for polymorphic variants and alleles of TC-Gα14. This selectionmay be achieved by subtracting out antibodies that cross-react withTC-Gα14 molecules from other species. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

III. Assays for taste modulation

A. Assays for taste cell specific G-protein alpha subunit activity

TC-Gα14 and its alleles, interspecies homologs, and polymorphic variantsparticipate in taste transduction. The activity of TC-Gα14 polypeptides,domains thereof, or fusion proteins of a TC-Gα14 polypeptide or a domainthereof can be assessed using a variety of in vitro and in vivo assaysthat measure functional, chemical and physical effects, e.g., measuringligand binding (e.g., radioactive ligand or GTP binding), signaltransduction enzyme activity (e.g., adenylate cyclase or phospholipaseC), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux,phosphorylation levels, transcription levels, neurotransmitter levels,and the like. Furthermore, such assays can be used to screen foractivators, inhibitors, and modulators of TC-Gα14. Such activators,inhibitors, and modulators of taste transduction activity are useful forcustomizing taste.

The TC-Gα14 of the assay will be selected from a polypeptide having asequence of SEQ ID NO:2 or alleles, orthologs, polymorphic variants, andconservatively modified variant thereof, e.g., from humans, rats, ormice. Alternatively, the TC-Gα14 of the assay will be derived from aeukaryote and include an amino acid subsequence having at least about70% amino acid sequence identity SEQ ID NO:2. Generally, the amino acidsequence identity will be at least 70%, optionally at least 75%, 80%,85%, optionally at least 90-95%. Optionally, the polypeptide of theassays will comprise a domain of TC-Gα14, such as a ligand bindingdomain, subunit association domain, active site, and the like. EitherTC-Gα14 or a domain thereof can be covalently linked to a heterologousprotein to create a chimeric protein used in the assays describedherein.

Modulators of TC-Gα144 activity are tested using TC-Gα14 polypeptides,chimeras or fragments thereof, as described above, either recombinant ornaturally occurring. The protein can be isolated, expressed in a cell,expressed in a membrane derived from a cell, expressed in tissue or inan animal, in any case either recombinant or naturally occurring. Forexample, tongue slices, dissociated cells from a tongue, transformedcells, cell membranes, or lipid bilayers can be used. Modulation istested using one of the in vitro or in vivo assays described herein.Taste transduction can also be examined in vitro with soluble or solidstate reactions, using a chimeric molecule, comprising, e.g., a ligandbinding domain of TC-Gα144, or a domain of TC-Gα14, or a full-lengthTC-Gα14. Furthermore, ligand-binding domains of the protein of interestcan be used in vitro in soluble or solid state reactions to assay forligand binding.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described herein. Anysuitable physiological change that affects TC-Gα14 activity can be usedto assess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3, cGMP, orcAMP.

Samples or assays that are treated with a test compound whichpotentially activates, inhibits, or modulates TC-Gα14 are compared tocontrol samples that are not treated without the test compound, toexamine the extent of modulation. Control samples (untreated withactivators, inhibitors, or modulators) are assigned a relative TC-Gα14activity value of 100%. Inhibition of TC-Gα14 is achieved when theTC-Gα14 activity value relative to the control is about 90% (e.g., 10%less than the control), preferably 50%, more preferably 25-0%.Activation of TC-Gα14 is achieved when the TC-Gα14 activity valuerelative to the control is 110% (e.g., 10% more than the control), morepreferably 150%, more preferably 200-500%, more preferably 1000-2000%.

In one embodiment, ligand binding to TC-Gα14, a domain, or chimericprotein can be tested in solution, in a bilayer membrane, attached to asolid phase, in a lipid monolayer, or in vesicles. Binding of amodulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index)hydrodynamic (e.g., shape), chromatographic, or solubility properties.In one example, radiolabeled GTP is used.

In another embodiment, receptor-G protein interactions are examined. Forexample, binding of a G protein comprising TC-Gα14 to a receptor or itsrelease from the receptor can be examined. For example, in the absenceof GTP, an activator will lead to the formation of a tight complex of aG protein (all three subunits) with the receptor. This complex can bedetected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors. An activator can be added to thereceptor and G protein in the absence of GTP, forming a tight complex,and then screen for inhibitors can be performed by looking atdissociation of the receptor-G protein complex. In the presence of GTP,release of the alpha subunit of the G protein from the other two Gprotein subunits serves as a criterion of activation.

In another example, activated GPCR receptors become substrates forkinases that phosphorylate the C-terminal tail of the receptor (andpossibly other sites as well). Thus, activators will promote thetransfer of ³²P from gamma-labeled GTP to the receptor, which can beassayed with a scintillation counter. The phosphorylation of theC-terminal tail will promote the binding of arrestin-like proteins andwill interfere with the binding of G proteins. The kinase/arrestinpathway plays a key role in the desensitization of many GPCR receptors.For example, compounds that modulate the duration a taste receptor staysactive would be useful as a means of prolonging a desired taste orcutting off an unpleasant one. For a general review of GPCR signaltransduction and methods of assaying signal transduction, see, e.g.,Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983);Bourne et al., Nature 10:349:117-27 (1991); Bourne et al., Nature348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92 (1998).

In another embodiment, signal transduction enzymes and second messengersare examined. An activated or inhibited G protein will in turn alter theproperties of target enzymes, channels, and other effector proteins. Theclassic examples are the activation of cGMP phosphodiesterase bytransducin in the visual system, adenylate cyclase by the stimulatory Gprotein, phospholipase C by Gq and other cognate G proteins, andmodulation of diverse channels by Gi and other G proteins. Downstreamconsequences can also be examined such as generation of diacyl glyceroland IP3 by phospholipase C, and in turn, for calcium mobilization byIP3.

Signal transduction typically initiates subsequent intracellular eventsvia, e.g., G-proteins and/or other enzymes, such as adenylate cyclase orphospholipase C, which are downstream from the G-proteins in tastetransduction pathways. For example, receptor activation and signaltransduction may result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase by G-protein α and βγ subunits. Theseintracellular cyclic nucleotides, in turn, may modulate other molecules,such as cyclic nucleotide-gated ion channels, e.g., channels that aremade permeable to cations by binding of cAMP or cGMP such as e.g., rodphotoreceptor cell channels and olfactory neuron channels (see, e.g.,Altenhofen et al., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) andDhallan et al., Nature 347:184-187 (1990)). In cases where activation ofTC-Gα14 results in a decrease in cyclic nucleotide levels, it may bepreferable to expose the cells to agents that increase intracellularcyclic nucleotide levels, e.g., forskolin, prior to adding a modulatorycompound to the cells in the assay. Cells for this type of assay can bemade by co-transfection of a host cell with DNA encoding a cyclicnucleotide-gated ion channel, DNA encoding TC-Gα14, DNA encoding a GPCRphosphatase and DNA encoding a G-protein coupled receptor (e.g.,metabotropic glutamate receptors, muscarinic acetylcholine receptors,dopanine receptors, serotonin receptors, and the like), which, whenactivated, causes a change in cyclic nucleotide levels in the cytoplasm.

In response to external stimuli, certain G-protein coupled receptors mayactivate an effector such as phospholipase C, through G-proteins.Activation of phospholipase C results in the production of inositol 1,4, 5-triphosphate (IP₃) and diacylglycerol (DAG) from inositol4,5-biphosphate PIP2) (Berridge & Irvine, Nature 312:315-21 (1984)). IP₃in turn stimulates the release of intracellular calcium ion stores.Cells may exhibit increased cytoplasmic calcium levels as a result ofcontribution from both intracellular stores and via activation of ionchannels, in which case it may be desirable although not necessary toconduct such assays in calcium-free buffer, optionally supplemented witha chelating agent such as EGTA, to distinguish fluorescence responseresulting from calcium release from internal stores. Thus, a change inthe level of second messengers, such as IP₃, DAG, or Ca²⁺ can be used toassess TC-Gα14 function. Furthermore, a change in the level of thesesecond messengers can be used to screen for activators, inhibitors, andmodulators of TC-Gα14 polypeptides.

For example, the activity of TC-Gα14 polypeptides can be assessed bymeasuring, e.g., changes in intracellular second messengers, such ascAMP, cGMP, IP₃, DAG, or Ca²⁺. Therefore, the second messenger levelscan be used as reporters for potential activators, inhibitors, andmodulators of TC-Gα14 polypeptides. In one embodiment, the changes inintracellular cAMP or cGMP can be measured using immunoassays. Themethod described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180(1995) may be used to determine the level of cAMP. Also, the methoddescribed in Felley-Bosco et al, Am. J. Resp. Cell and Mol. Biol.11:159-164 (1994) may be used to determine the level of cGMP. Further,an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat.No. 4,115,538, herein incorporated by reference.

In another example, the second messenger phosphatidyl inositol (PI)hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, hereinincorporated by reference. Briefly, the assay involves labeling of cellswith ³H-myoinositol for 48 or more hrs. The labeled cells are treatedwith a test compound for one hour. The treated cells are lysed andextracted in chloroform-methanol-water after which the inositolphosphates were separated by ion exchange chromatography and quantifiedby scintillation counting. Fold stimulation is determined by calculatingthe ratio of cpm in the presence of agonist to cpm in the presence ofbuffer control. Likewise, fold inhibition is determined by calculatingthe ratio of cpm in the presence of antagonist to cpm in the presence ofbuffer control (which may or may not contain an agonist).

In another example, intracellular Ca²⁺ levels can be analyzed, e.g.,using fluorescent Ca²⁺ indicator dyes and fluorometric imaging (see,e.g., Hall et al., Nature 331:729 (1988); Kudo et al., Neuros.50:619-625 (1992); van Heugten et al., J. Mol. Cell. Cardiol. 26:1081-93(1994)).

In another embodiment, the activity of TC-Gα14 can also be assessed bymeasuring changes in ion flux. Changes in ion flux may be measured bydetermining changes in polarization (i.e., electrical potential) of thecell or membrane expressing TC-Gα14. One means to determine changes incellular polarization is by measuring changes in current (therebymeasuring changes in polarization) with voltage-clamp and patch-clamptechniques, e.g., the “cell-attached” mode, the “inside-out” mode, andthe “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med.336:1575-1595 (1997)). Whole cell currents are conveniently determinedusing the standard methodology (see, e.g., Hamil et al., PFlugers.Archiv. 391:85 (1981)). Other known assays include: radiolabeled ionflux assays and fluorescence assays using voltage-sensitive dyes (see,e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988);Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J.Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. MembraneBiology 137:59-70 (1994)). A method for the whole-cell recording fromnon-dissociated taste cells within mouse taste bud is described inMiyamoto et al., J. Neurosci Methods 64:245-252 (1996). Therefore,changes in ion flux can be used to screen for activators, inhibitors,and modulators of TC-Gα14. Generally, the compounds to be tested arepresent in the range from 1 pM to 100 mM.

Assays for measuring changes in ion flux include cells that are loadedwith ion or voltage sensitive dyes to report TC-Gα14 activity. Assaysfor determining activity of these polypeptides can also use knownagonists and antagonists for these polypeptides as negative or positivecontrols to assess activity of tested compounds. In assays foridentifying modulatory compounds (e.g., agonists, antagonists), changesin the level of ions in the cytoplasm or membrane voltage will bemonitored using an ion sensitive or membrane voltage fluorescentindicator, respectively. Among the ion-sensitive indicators and voltageprobes that may be employed are those disclosed in the Molecular Probes1997 Catalog.

In another embodiment, phosphorylation of taste cell specific proteinscan be measured to assess the effects of a test compound on TC-Gα14function. This can be achieved by using a method disclosed in, e.g.,U.S. Pat. No. 5,834,216, herein incorporated by reference. A duplicatecell culture containing expressed TC-Gα14 can be prepared. One of theduplicate cultures is exposed to a test compound. Cell lysates from theduplicate cultures are prepared. The cell lysates are contacted with ATPor a GTP, wherein the nucleotide has a gamma-phosphate having adetectable label, or an analog of a gamma phosphate (i.e., having alabel capable of being transferred to a phosphorylation site such asgamma S³⁵). The level of phosphorylated taste cell specific proteins maybe measured by precipitating the cell lysates with an antibody specificfor taste cell specific proteins. After precipitation, phosphorylated(labeled) taste cell specific proteins may be separated from othercellular proteins by electrophoresis or by chromatographic methods. Byway of example, labeled taste cell specific proteins may be separated ondenaturing polyacrylamide gels after which the separated proteins may betransferred to, for example, a nylon or nitrocellulose membrane followedby exposure to X-ray film. Relative levels of phosphorylation are thendetermined after developing the exposed X-ray film and quantifying thedensity of bands corresponding to the taste cell specific proteins, forexample, densitometry. The autoradiograph may also be used to localizethe bands on the membrane corresponding to labeled taste cell specificproteins after which they may be excised from the membrane and countedby liquid scintillation or other counting methods. Using this method, atest compound which effects the function of TC-Gα14 is identified by itsability to increase or decrease phosphorylation of taste cell specificproteins compared to control cells not exposed to the test compound.

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on TC-Gα14 function. A host cellcontaining TC-Gα14 is contacted with a test compound for a sufficienttime to effect any interactions, and then the level of TC-Gα14 geneexpression is measured. The amount of time to effect such interactionsmay be empirically determined, such as by running a time course andmeasuring the level of transcription as a function of time. The amountof transcription may be measured by using any method known to those ofskill in the art to be suitable. For example, mRNA expression of TC-Gα14may be detected using northern blots or their polypeptide products maybe identified using immunoassays. Alternatively, transcription basedassays using reporter gene may be used as described in U.S. Pat. No.5,436,128, herein incorporated by reference. The reporter genes can be,e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, P-galactosidase and alkaline phosphatase. Furthermore,TC-Gα14 can be used as indirect reporters via attachment to a secondreporter such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks TC-Gα14. A substantiallyidentical cell may be derived from the same cells from which therecombinant cell was prepared but which had not been modified byintroduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of TC-Gα14.

Other physiological change that affects TC-Gα14 activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. For example, the influence of a test compound on the GTPaseactivity of TC-Gα14 can be assessed using the method described in U.S.Pat. No. 5,817,759, which patent is incorporated herein by reference.When the functional consequences are determined using intact cells oranimals, one can also measure a variety of effects such as transmitterrelease, hormone release, transcriptional changes to both known anduncharacterized genetic markers (e.g., northern blots), changes in cellmetabolism such as cell growth or pH changes, and the like.

In a preferred embodiment, TC-Gα14 activity is measured by expressingTC-Gα14 in a heterologous cell with a TC-GPCR (see U.S. Ser. No.09/361,652, filed Jul. 27, 1999 and U.S. Ser. No. 09/361,631, filed Jul.27, 1999, now U.S. Pat. No. 6,383,778). Exemplary polynucleotidesequences encoding rat, mouse, and human GPCR-B3 are presented in SEQ IDNOs:3-5, respectively, encoding polypeptides having the amino acidsequences of SEQ ID NOs:6-8. Exemplary polynucleotide sequences encodingrat, mouse, and human GPCR-B4 are presented in SEQ ID NOs:9-11,respectively, encoding polypeptides having the amino acid sequences ofSEQ ID NOs: 12-14. As shown in Example I below, TC-Gα14 is specificallyexpressed in taste receptor cells, and also co-expressed with GPCR1-B3and GPCR-B4, in different taste papillae. As described above, HEK-293cells may be used as a heterologous host cell, and modulation of tastetransduction is assayed by measuring changes in intracellular Ca²⁺levels.

B. Modulators

The compounds tested as modulators of TC-Gα14 can be any small chemicalcompound, or a biological entity, such as a protein, sugar, nucleic acidor lipid. Alternatively, modulators can be genetically altered versionsof TC-Gα14. Typically, test compounds will be small chemical moleculesand peptides. Essentially any chemical compound can be used as apotential modulator or ligand in the assays of the invention, althoughmost often compounds can be dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al, Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., _(J. Amer. Chem. Soc.) 116:2661(1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/orpeptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)),nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S.Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.5,549,974; pyrrolidines, U.S. Pat. No. 5,525,735 and U.S. Pat. No.5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,PA, Martek Biosciences, Columbia, Md., etc.).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingTC-Gα14 is attached to a solid phase substrate. In the high throughputassays of the invention, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to run a separate assay against aselected potential modulator, or, if concentration or incubation timeeffects are to be observed, every 5-10 wells can test a singlemodulator. Thus, a single standard microtiter plate can assay about 100(e.g., 96) modulators. If 1536 well plates are used, then a single platecan easily assay from about 100- about 1500 different compounds. It ispossible to assay several different plates per day; assay screens for upto about 6,000-20,000 different compounds is possible using theintegrated systems of the invention. More recently, microfluidicapproaches to reagent manipulation have been developed.

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provide soluble assays using moleculessuch as a domain such as ligand binding domain, an active site, asubunit association region, etc.; a domain that is covalently linked toa heterologous protein to create a chimeric molecule; TC-Gα14; a cell ortissue expressing TC-Gα14, either naturally occurring or recombinant. Inanother embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the domain, chimeric molecule,TC-Gα14, or cell or tissue expressing TC-Gα14 is attached to a solidphase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

D. Computer-Based Assays

Yet another assay for compounds that modulate TC-Gα14 activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of TC-Gα14 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering G-protein amino acid sequences of at least 10 amino acidresidues or corresponding nucleic acid sequences encoding a TC-Gα14polypeptide into the computer system. The amino acid sequence of thepolypeptide of the nucleic acid encoding the polypeptide is selectedfrom the group consisting of SEQ ID NO:2 and conservatively modifiedversions thereof. The amino acid sequence represents the primarysequence or subsequence of the protein, which encodes the structuralinformation of the protein. At least 10 residues of the amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art. Thethree-dimensional structural model of the protein can be saved to acomputer readable form and be used for further analysis (e.g.,identifying potential ligand binding regions of the protein andscreening for mutations, alleles and interspecies homologs of the gene).

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the TC-Gα14 protein to identify ligands that bind to TC-Gα14.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein. The results, such as three-dimensionalstructures for potential ligands and binding affinity of ligands, canalso be saved to a computer readable form and can be used for furtheranalysis (e.g., generating a three dimensional model of mutated proteinshaving an altered binding affinity for a ligand).

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of TC-Gα14 genes. Suchmutations can be associated with disease states or genetic traits. Asdescribed above, high density oligonucleotide arrays (GeneChip™) andrelated technology can also be used to screen for mutations, polymorphicvariants, alleles and interspecies homologs. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes. Identification of the mutated TC-Gα14 genes involvesreceiving input of a first nucleic acid or amino acid sequence encodingTC-Gα14, selected from the group consisting of SEQ ID NO:1, or SEQ IDNO:2, and conservatively modified versions thereof. The sequence isentered into the computer system as described above and then saved to acomputer readable form. The first nucleic acid or amino acid sequence isthen compared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in TC-Gα14 genes, and mutations associatedwith disease states and genetic traits.

III. Isolation of the Nucleic Acid Encoding TC-Gα14

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingTC-Gα14

In general, the nucleic acid sequences encoding TC-Gα14 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries by hybridization with a probe, or isolated using amplificationtechniques with oligonucleotide primers. For example, TC-Gα14 sequencesare typically isolated from mammalian nucleic acid (genomic or cDNA)libraries by hybridizing with a nucleic acid probe, the sequence ofwhich can be derived from SEQ ID NO: 1. The mouse sequence of SEQ ID NO:1 can be used to isolate orthologs from other species, such as human andrat. A suitable tissue from which TC-Gα14 RNA and cDNA can be isolatedis tongue tissue, preferably taste bud tissue, more preferablyindividual taste cells. For example, circumvallate, foliate, fungiformtaste receptor cells can be used to isolate TC-Gα14 RNA and cDNA.

Amplification techniques using primers can also be used to amplify andisolate TC-Gα14 from DNA or RNA (see, e.g., Dieffenfach & Dveksler, PCRPrimer: A Laboratory Manual (1995)). These primers can be used, e.g., toamplify either the full length sequence or a probe of one to severalhundred nucleotides, which is then used to screen a mammalian libraryfor full-length TC-Gα14.

Nucleic acids encoding TC-Gα14 can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:2.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to TC-Gα14 can be isolated using TC-Gα14 nucleicacid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone TC-Gα14, and its polymorphic variants, alleles, andinterspecies homologs, by detecting expressed homologs immunologicallywith antisera or purified antibodies made against TC-Gα14, which alsorecognize and selectively bind to the TC-Gα14 homolog.

To make a cDNA library, one should choose a source that is rich inTC-Gα14 mRNA, e.g., tongue tissue, or isolated taste buds. The mRNA isthen made into cDNA using reverse transcriptase, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Nail.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating TC-Gα14 nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and U.S. Pat.No. 4,683,202; PCR Protocols. A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of TC-Gα14 directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify TC-Gα14 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of TC-Gα14 encoding mRNA in physiologicalsamples, for nucleic acid sequencing, or for other purposes. Genesamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

Gene expression of TC-Gα14 can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, and the like. In oneembodiment, high density oligonucleotide arrays technology (e.g.,GeneChip™) is used to identify homologs and polymorphic variants of theTC-Gα14 of the invention (see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14:869-876 (1998); Kozal et al., Nat. Med. 2:753-759(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998)).

Synthetic oligonucleotides can be used to construct recombinant TC-Gα14genes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and non-sense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the TC-Gα14 nucleic acid. Thespecific subsequence is then ligated into an expression vector.

Optionally, nucleic acids encoding chimeric proteins comprising TC-Gα14or domains thereof can be made according to standard techniques. Forexample, a domain such as ligand binding domain, an active site, asubunit association region, a membrane binding domain etc., can becovalently linked to a heterologous protein. Heterologous proteins ofchoice include, e.g., green fluorescent protein, β-gal, glutamatereceptor, and the rhodopsin presequence.

The nucleic acid encoding TC-Gα14 is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding TC-Gα14, one typically subclones TC-Gα14 into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the TC-Gα14 proteins areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the TC-Gα14 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding TC-Gα14and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding TC-Gα14 may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a TC-Gα14 encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of TC-Gα14proteins, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983)).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingTC-Gα14.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofTC-Gα14, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of TC-Gα14

Either naturally occurring or recombinant TC-Gα14 can be purified foruse in functional assays. Preferably, recombinant TC-Gα14 is purified.Naturally occurring TC-Gα14 is purified, e.g., from mammalian tissuesuch as tongue tissue, and any other source of a TC-Ga4 homolog.Recombinant TC-Gα14 is purified from any suitable expression system.

TC-Gα14 may be purified to substantial purity by standard techniques,including selective precipitation with such substances as ammoniumsulfate; column chromatography, immunopurification methods, and others(see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant TC-Gα14 is beingpurified. For example, proteins having established molecular adhesionproperties can be reversible fused to TC-Gα14. With the appropriateligand, TC-Gα14 can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally TC-Gα14 could be purifiedusing immunoaffinity columns.

A. Purification of TC-Gα14 from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of TC-Gα14inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-formin G-proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing reformation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. TC-Gα14 is separated fromother bacterial proteins by standard separation techniques, e.g., withNi-NTA agarose resin.

Alternatively, it is possible to purify TC-Gα14 from bacteria periplasm.After lysis of the bacteria, when TC-Gα14 is exported into the periplasmof the bacteria, the periplasmic fraction of the bacteria can beisolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

B. Standard Protein Separation Techniques for Purifying TC-Gα14

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of TC-Gα14 can be used to isolate them fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

TC-Gα14 can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified. All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of TC-Gα14

In addition to the detection of TC-Gα14 genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect TC-Gα14, e.g., to identify taste receptor cells and variants ofTC-Gα14. Immunoassays can be used to qualitatively or quantitativelyanalyze TC-Gα14. A general overview of the applicable technology can befound in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to TC-Gα14

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with TC-Gα14 are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of TC-Gα14 comprising immunogens may be used to produceantibodies specifically reactive with TC-Gα14. For example, recombinantTC-Gα14 or an antigenic fragment thereof, is isolated as describedherein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring G-protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to TC-Gα14. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-TC-Gα14proteins or even other related proteins from other organisms, using acompetitive binding immunoassay. Specific polyclonal antisera andmonoclonal antibodies will usually bind with a K_(d) of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

Once TC-Gα14 specific antibodies are available, TC-Gα14 can be detectedby a variety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

B. Immunological Binding Assays

TC-Gα14 can be detected and/or quantified using any of a number of wellrecognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and U.S. Pat. No. 4,837,168). For areview of the general immunoassays, see also, Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunologicalbinding assays (or immunoassays) typically use an antibody thatspecifically binds to a protein or antigen of choice (in this case theTC-Gα14, or antigenic subsequence thereof). The antibody (e.g.,anti-TC-Gα14) may be produced by any of a number of means well known tothose of skill in the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled polypeptide ofTC-Gα14 or a labeled anti-TC-Gα14 antibody. Alternatively, the labelingagent may be a third moiety, such a secondary antibody, thatspecifically binds to the antibody/TC-Gα14 complex (a secondary antibodyis typically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting TC-Gα14 in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-TC-Gα14 antibodies can be bound directly toa solid substrate on which they are immobilized. These immobilizedantibodies then capture TC-Gα14 present in the test sample. TC-Gα14 isthus immobilized is then bound by a labeling agent, such as a secondTC-Gα14 antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of TC-Gα14 present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) TC-Gα14 displaced (competed away) from an anti-TC-Gα14antibody by the unknown TC-Gα14 present in a sample. In one competitiveassay, a known amount of TC-Gα14 is added to a sample and the sample isthen contacted with an antibody that specifically binds to TC-Gα14. Theamount of exogenous TC-Gα14 bound to the antibody is inverselyproportional to the concentration of TC-Gα14 present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of TC-Gα14 bound to the antibody may bedetermined either by measuring the amount of TC-Gα14 present in aTC-Gα14/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of TC-Gα14 may be detected byproviding a labeled TC-Gα14 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known TC-Gα14, is immobilized on a solid substrate. Aknown amount of anti-TC-Gα14 antibody is added to the sample, and thesample is then contacted with the immobilized TC-Gα14. The amount ofanti-TC-Gα14 antibody bound to the known immobilized TC-Gα14 isinversely proportional to the amount of TC-Gα14 present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2 can be immobilized to a solid support.Proteins (e.g., TC-Gα14 proteins and homologs) are added to the assaythat compete for binding of the antisera to the immobilized antigen. Theability of the added proteins to compete for binding of the antisera tothe immobilized protein is compared to the ability of TC-Gα14 encoded bySEQ ID NO:2 to compete with itself The percent crossreactivity for theabove proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the addedproteins listed above are selected and pooled. The cross-reactingantibodies are optionally removed from the pooled antisera byimmunoabsorption with the added considered proteins, e.g., distantlyrelated homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of TC-Gα14 to theimmunogen protein (i.e., TC-Gα14 of SEQ ID NO:2). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to a TC-Gα14immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of TC-Gα14 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind TC-Gα14. The anti-TC-Gα14 antibodies specificallybind to TC-Gα14 on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-TC-Gα14 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize TC-Gα14, orsecondary antibodies that recognize anti-TC-Gα14.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VIII. Kits

TC-Gα14 and its homologs are a useful tool for identifying tastereceptor cells, for forensics and paternity determinations, and forexamining taste transduction (e.g., generating a topographical mapbetween the taste cells of the tongue and the corresponding tastecenters in the brain). Specific reagents that specifically hybridize toTC-Gα14 nucleic acid, such as its probes and primers, and specificreagents that specifically bind to the TC-Gα14 protein, e.g., itsantibodies are used to examine taste cell expression and tastetransduction regulation.

Nucleic acid assays for the presence of TC-Gα14 DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art, suchas Southern analysis, northern analysis, dot blots, RNase protection,high density oligonucleotide arrays, S1 analysis, amplificationtechniques such as PCR and LCR, and in situ hybridization. In in situhybridization, for example, the target nucleic acid is liberated fromits cellular surroundings in such as to be available for hybridizationwithin the cell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization; Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987). In addition, TC-Gα14 protein can be detectedwith the various immunoassay techniques described above. The test sampleis typically compared to both a positive control (e.g., a sampleexpressing recombinant TC-Gα14) and a negative control.

The present invention also provides for kits for screening formodulators of TC-Gα14. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: TC-Gα14, reaction tubes, andinstructions for testing TC-Gα14 activity. Preferably, the kit containsbiologically active TC-Gα14. A wide variety of kits and components canbe prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

IX. Administration and Pharmaceutical Compositions

Taste modulators can be administered directly to the mammalian subjectfor modulation of taste in vivo. Administration is by any of the routesnormally used for introducing a modulator compound into ultimate contactwith the tissue to be treated, preferably the tongue or mouth. The tastemodulators are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch modulators are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington 's Pharmaceutical Sciences,17^(th) ed. 1985)).

The taste modulators, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by orally, topically, intravenously,intraperitoneally, intravesically or intrathecally. Preferably, thecompositions are administered orally or nasally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. The modulators can also be administered as part a of preparedfood or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular taste modulators employed and the condition of the subject,as well as the body weight or surface area of the area to be treated.The size of the dose also will be determined by the existence, nature,and extent of any adverse side-effects that accompany the administrationDf a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administeredin a physician may evaluate circulating plasma levels of the modulator,modulator toxicities, and the production of anti-modulator antibodies.In general, the dose equivalent of a modulator is from about 1 ng/kg to10 mg/kg for a typical subject.

For administration, taste modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Taste Receptor Cell Specific Expression of TC-Gα14

Clones representing different Ga subunits were amplified by PCR andlabeled for in situ hybridizations with tongue sections. These includeGα_(s), Gα_(i1), Gα_(i2), Gα_(i3), Gα_(z), Gα_(t1), Gα_(t2), Gα_(olf),Gα_(q), Gα₁₁, Gα₁₂, Gα₁₄ and Gα₁₅ (see Simon et al., Science 252:802-8(1991)). Gα₁₄ amino acid sequence is published in Wilkie et al. PNAS USA88:10049-10053 (1991). Taste tissue was obtained from adult rats andmice. No sex-specific differences in expression patterns were observed,therefore male and female animals were used interchangeably. For foliatesections, no differences in expression pattern were observed between thepapillae. Fresh frozen sections (14 μm) were attached to silanizedslides and prepared for in situ hybridization as described previously(Ryba & Tirindelli, Neuron 19:371-379 (1997)). All in situhybridizations were carried out at high stringency (5×SSC, 50%formamide, 72° C.). For single-label detection, signals were developedusing alkaline-phosphatase conjugated antibodies to digoxigenin andstandard chromogenic substrates (Boehringer Mannheim). For double-labelfluorescent detection, an alkaline-phosphatase conjugatedanti-fluorescein antibody (Amersham) and a horse-peroxidase conjugatedanti-digoxigenin antibody were used in combination with fast-red andtyramide fluorogenic substrates (Boehringer Mannheim and New EnglandNuclear).

These experiments demonstrate that Gα14 is specifically and selectivelyexpressed in circumvallate, foliate and fungiform taste receptor cellsof the tongue, as shown by in situ hybridization. Therefore, Gα14 is a Galpha subunit that is specifically expressed in taste receptor cells.Furthermore, this gene is co-expressed with both GPCR-B3 and GPCR-B4receptors in the different taste papillae (see U.S. Ser. No. 09/361,652,filed Jul. 27, 1999 and U.S. Ser. No. 09/361,631, filed Jul. 27, 1999,now U.S. Pat. No. 6,383,778). Exemplary polynucleotide sequencesencoding rat, mouse, and human GPCR-B3 are presented in SEQ ID NOs:3-5,respectively, encoding polypeptides having the amino acid sequences ofSEQ ID NOs:6-8. Exemplary polynucleotide sequences encoding rat, mouse,and human GPCR-B4 are presented in SEQ ID NOs:9-11, respectively,encoding polypeptides having the amino acid sequences of SEQ IDNOs:12-14.

Example II Expression of TC-Gα in a Heterologous Cell with a Taste CellSpecific G-Protein Coupled Receptor

TC-Gα14 is expressed in a heterologous cell with a taste cell specificG-protein coupled receptor such as GPCR-B3 or GPCR-B4 to screen foractivators, inhibitors, and modulators of TC-Gα14. Modulation of tastetransduction is assayed by measuring changes in intracellular Ca²⁺levels, which change in response to modulation of the TC-Gα14 signaltransduction pathway via administration of a molecule that associateswith TC-Gα14. Changes in Ca²⁺ levels are preferably measured usingfluorescent Ca²⁺ indicator dyes and fluorometric imaging. The amount of[Ca²+]_(i) is then compared to the amount of [Ca²⁺]_(i) in either thesame cell in the absence of the test compound, or it may be compared tothe amount of [Ca²⁺]_(i) in a substantially identical cell that lacksTC-Gα14.

SEQUENCE LISTING Mouse TC-Gα14 nucleotide sequence (AccessionM80631)-SEQ ID NO:1   1 aactgccttc gagaagcgtt agcctagaga tccgagcctcttctccatac catagttggt  61 tcaggtggtt tcctcttcaa accttgcgtc tgcggataatccgcgcggcc gggcgttaag  121 ctccaggtcc ctgtcgctcc gtcgaggtgg caagccatggccggctgctg ctgtttgtct  181 gcggaggaga aagagtctca gcgcatcagc gcggagatcgagcggcacgt tcgccgcgac  241 aagaaggacg cgcgccggga gctcaagctg ctgttgctgggaaccggtga gagtgggaaa  301 agcaccttta tcaagcagat gaggataatc catgggtctggctacagtga tgaagataga  361 aagggcttca cgaagctggt ttaccaaaac atattcacggccatgcaagc catgatcaga  421 gcaatggata ccctgaggat acaatacatg tgtgagcagaataaggaaaa tgcccagatc  481 atcagggaag tggaagtaga caaggtcact gcactctctagagaccaggt ggcagccatc  541 aagcagctgt ggctggatcc cggaatccag gagtgttacgacaggaggag ggagtaccag  601 ctgtcagact ctgccaaata ttacctgacg gacattgagcgtatcgccat gccctctttc  661 gtgccaacac aacaggatgt gcttcgtgtt agagtgcccaccactggcat catagaatat  721 ccattcgacc tggaaaacat catcttccga atggtggatgttggtggcca gcgatctgaa  781 cgacggaaat ggattcactg ctttgagagt gtcacctccatcattttctt ggttgctctg  841 agtgaatatg accaggttct ggctgagtgt gacaatgagaaccgcatgga ggagagcaaa  901 gccctgttta gaaccatcat cacctacccc tggtttctgaactcctccgt gattctgttc  961 ttaaacaaga aggatcttct agaggagaaa atcatgtactctcatctaat tagctacttc 1021 ccagagtaca caggaccaaa gcaagatgtc aaagcggccagggactttat cctgaagctg 1081 tatcaagacc agaatcctga caaagagaag gttatctattctcacttcac ttgtgctaca 1141 gacaccgaga atatccgctt tgtgtttgct gctgtcaaagacacaatcct acagctaaac 1201 ctacgggagt tcaacttggt gtaaatggag ggcctactcctccgagacag agggtgatct 1261 gagcccttcc tgcctgatct acaagtgctt ctggaccaggacctaaggac attatgtagc 1321 ccacaggaca gagatgggta gtgcaatgtg aaaaatacttcaccaaccct tttaagtgtc 1381 tttaattctt cactgtclaa ctcttttctc gccttttggttgaacgatta ggtatcattt 1441 ttgagtggtt ccccctctcc tattttttta aactagtgttcaacagttat taaaaaatca 1501 tgc// Mouse TC-Gα14 amino acid sequence(Accession 193569)-SEQ ID NO:2   1 magccclsae ekesqrisae ierhvrrdkkdarrelklll lgtgesgkst fikqmriihg  61 sgysdedrkg ftklvyqnif tamqamiramdtlriqymce qnkenaqiir evevdkvtal  121 srdqvaaikq lwldpgiqec ydrrreyqlsdsakyyltdi eriampsfvp tqqdvlrvrv  181 pttgiieypf dleniifrmv dvggqrserrkwihcfesvt siiflvalse ydqvlaecdn  241 enrmeeskal frtiitypwf lnssvilflnkkdlleekim yshlisyfpe ytgpkqdvka  301 ardfilklyq dqnpdkekvi yshftcatdtenirfvfaav kdtilqlnlr efnlv

1. A method for identifying a compound that modulates signaltransduction in taste cells, the method comprising the steps of: (i)contacting a cell which expresses a taste cell specific G-protein alphasubunit polypeptide and a taste cell specific G protein coupled receptorwith the compound, the G-protein alpha subunit polypeptide comprising asequence of SEQ ID NO:2; wherein the G-protein alpha subunit polypeptideis a subunit of a heterotrimeric G-protein which binds GTP and theG-protein alpha subunit polypeptide is recombinantly expressed in thecell, wherein the taste cell specific G protein coupled receptorcomprises the amino acid sequence selected from the group consisting ofSEQ ID NOs. 12, 13, and 14; and (ii) determining a functional effect ofthe compound upon the cell expressing the taste cell specific G-proteinalpha subunit polypeptide and the taste cell specific G protein coupledreceptor, thereby identifying a compound that modulates signaltransduction in taste cells.
 2. The method of claim 1, wherein thefunctional effect is a chemical effect.
 3. The method of claim 1,wherein the functional effect is determined by measuring increased ordecreased binding of radiolabeled GTP to the G-protein alpha subunitpolypeptide or to a G protein comprising the G-protein alpha subunitpolypeptide.
 4. The method of claim 1, wherein the G-protein alphasubunit polypeptide is from a mouse, a rat or a human.